Cellular interactions are key to many events in vascular biology. Cell surface adhesion molecules mediate many of the interactions between leukocytes, platelets and the vessel wall. In response to inflammatory stimuli, leukocytes and platelets in the adjacent vasculature initially roll on the blood vessel wall, then stick, and finally transmigrate to the site of insult. The initial rolling event involves a class of adhesion proteins termed selectins (P-, E-, and L-selectin) which mediate the interaction between leukocytes, platelets and endothelial cells by their recognition of specific carbohydrate counter-structures, including sialyl-Lewis x. The primary sequence/motif structure of each of the selectins is similar. Each contains a N-terminal, 118-amino acid calcium-dependent lectin domain, an EGF motif, a variable number of tandem repetitive motifs related to motifs found in complement regulatory domains, a transmembrane domain and a short cytoplasmic tail.
P-selectin is a 140-kDa integral granule membrane glycoprotein localized to the xcex1-granules of platelets and the Weibel-Palade bodies of endothelial cells and is rapidly expressed on both cell types on cell activation. This suggests that endothelial P-selectin is a critical molecule mediating initial adhesion events in acute inflammation, a view recently supported by a number of in vivo inflammatory models including neutrophil-dependent acute lung injury (Mulligan et al. (1992) J. Clin. Invest. 90, 1600), endotoxin-induced neutropenia (Coughlan et al. (1994) J. Exp. Med. 179, 329), reperfusion injury (Asako et al. (1994) J. Clin. Invest. 93, 1508) and histamine-induced leukocyte rolling in post capillary venules (Weyrich et al. (1993) J. Clin. Invest. 91, 2620). P-selectin binds to 10,000-20,000 copies of a single class of binding sites on neutrophils and HL60 cells.
Sako et al. ((1993) Cell 75, 1179) have cloned a ligand for P-selectin, termed P-selectin glycoprotein ligand-1 (PSGL-1) found on the surface of leukocytes (see also copending application Ser. No. 08/316,305). PSGL-1 is a 220 kDa, disulfide-linked homodimeric sialomucin which, when expressed by recombinant methodology with the appropriate fucosyltransferase, binds P-selectin, E-selectin and L-selectin in a similar calcium-dependent manner to the PSGL-1 on neutrophils. PSGL-1 has a signal peptide sequence of 17 amino acids followed by a 24-amino acid PACE cleaved propeptide sequence. The mature N-terminus of PSGL-1 contains an unusual stretch of twenty amino acids which is rich in negatively-charged aspartate and glutamate residues and which contains three tyrosine residues which meet the consensus sequence for 0-sulfation by a golgi sulfotransferase. At least one of these tyrosine residues is sulfated as evaluated by site-directed mutagenesis (Sako et al.).
In addition to binding P-selectin, PSGL-1 also binds L- and E-selectin. In contrast to P-selectin, however, the requirements for E-selectin recognition are much less rigid. (Spertinit et al., J. Cell. Biol. 135:523 (1996)). E-selectin binds a wide variety of sialomucin structures if they co-express the sialyl-Lewis x structure. L-selectin binds to a number of different counter-receptors, GLYCAM-1, MadCAM-1 and CD34, which like PSGL-1, are also sialomucins. A major question currently unresolved is what determines selectin specificity in the recognition of specific counter-receptor structures. P-, E- and L-selectin are 60-70% homologous in their N-terminal, 118-amino acid lectin motifs and each similarly recognizes the sialyl-Lewis x and sialyl-Lewis a carbohydrate structures. Further, binding of P-selectin to its receptor on neutrophils is four to five orders of magnitude more avid than the binding of sialyl-Lewis x. While differences in specificity and avidity may in part be accounted for by either the presentation of multiple sialyl-Lewis carbohydrate structures on the receptor mucin core or by subtle differences in carbohydrate structure, it is probable that the protein component of the sialomucin also determines selectin interaction.
Although the inflammatory response mediated by the P-selectin/PSGL-1 interaction is a part of the body""s normal defense system, inappropriate inflammatory responses can also result in the development of various inflammatory disease states. It would, therefore, be desirable to provide agents for interfering with or blocking the selectin/PSGL-1 interaction in order to treat inflammatory disease.
GP1bxcex1 is a component of the glycoprotein (GP) Ib-IX complex found on the surface of platelets and serving as a receptor for von Willibrand factor (vWF). The interaction of the GP IB-IX complex with vWF mediates attachment of platelets to the blood vessel wall at the site of injury. It has also can cause aggregation of platelets in high shear conditions and enable platelet activation at low concentrations of thrombin.
Mocarhagin, a protease found in the venom of cobras (including the Mozambiquan spitting cobra, Naja mossambica mossambica, a.k.a. Naja mocambique mocambique), has been found to cleave PSGL-1, resulting in disruption of P- and L-selectin mediated cell adhesion. Preparations of mocarhagin have been reported and demonstrated to serve this purpose. See, U.S. Pat. No. 5,659,018; DeLuca et al., J. Biol. Chem. 270: 26734 (1995); Ward et al., Biochem. 35: 4929 (1996). (Spertini et al.)
In addition, it also has been reported that Mocarhagin is capable of cleaving GP1bxcex1 at a position proximal to sulfated tyrosine residues within the critical vWF binding domain and disrupting the binding activity of GP1bxcex1: DeLuca et al., J. Biol. Chem. 270: 26734 (1995); Dong et al., Biochemistry, 33: 13946 (1994).
It is therefore anticipated that an agent that can disrupt this interaction may have therapeutic application in a variety of thrombotic disorders such as restenosis and DVT.
However, applicants have discovered that the preparations described in these documents is only partially purified. Since it is necessary for mocarhagin proteins to be provided in highly purified form for such proteins to be used for therapeutic purposes, it would be desirable to provide highly purified preparations of mocarhagin proteins.
It would also be desirable to identify and isolate polynucleotides encoding mocarhagin proteins in order to produce such proteins by recombinant methods.
The present invention provides compositions comprising a mocarhagin protein at least 95% free of other cobra proteins (preferably 95% free of all other proteins). Preferably, the mocarhagin is homogeneous (i.e., free of other proteins). In preferred embodiments, the mocarhagin protein is full-length mocarhagin (as described below). In other embodiments, the mocarhagin protein is a fragment of full-length mocarhagin having mocarhagin proteolytic activity. Preferably, the mocarhagin protein is characterized by at least one characteristic selected from the group consisting of:
(a) a molecular weight of approximately 55 kDa under reducing conditions;
(b) a molecular weight of approximately 55 kDa under nonreducing conditions;
(c) an N-terminal amino acid sequence comprising
TNTPEQDRYLQAKKYIEFYVVVDNVMYRKY (SEQ ID NO:1);
(d) mocarhagin proteolytic activity;
(e) the ability to inhibit platelet binding to vWF;
(f) requirement of calcium ion for activity;
(g) requirement of zinc ion for activity;
(h) an activity substantially inhibited by excess EDTA; and
(i) an activity substantially inhibited by high concentrations of DFP.
In some embodiments, the mocarhagin protein has the N-terminal sequences TNTPEQDRYLQAKKYIEFYVVVDNVMYRKYTGKLHVITXXVYEMNALN (SEQ ID NO:2).
In particularly preferred embodiments, the mocarhagin protein is capable of cleaving capable of cleaving a material selected from the group consisting of anionic polypeptides containing sulfated tyrosine residues, PSGL-1 and GP1bxcex1. PSGL-1 and/or GP1bxcex1. Compositions comprising a therapeutically effective amount of a mocarhagin protein and a pharmaceutically acceptable carrier are also provided.
Methods of treating an inflammatory disease and thrombotic disorders and of inhibiting selectin-mediated binding comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a mocarhagin protein to a mammalian subject are disclosed.
The invention also provides a method of isolating mocarhagin from venom, said method comprising:
(a) subjecting a composition comprising cobra venom to a heparin affinity chromatography column;
(b) subjecting the eluate from said heparin affinity column to a size exclusion column;
(c) subjecting the eluate from said size exclusion column to a Mono S column; and
(d) eluting said mocarhagin from said Mono S column.
Compositions comprising a protein isolated according to these methods (and optionally further comprising a pharmaceutically acceptable carrier) are also encompassed by the claimed invention. Such compositions can also be used in methods of treating an inflammatory disease and of inhibiting selectin-mediated binding which comprise administering a therapeutically effective amount of such compositions to a mammalian subject.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 24 to amino acid 621;
(c) the amino acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid 621;
(d) fragments of the amino acid sequence of SEQ ID NO:6 encoding a protein having mocarhagin activity; and
(e) the amino acid sequence encoded by the cDNA insert of clone NMM-1 deposited under accession number ATCC 209588;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 78 to nucleotide 1940;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 147 to nucleotide 1940;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 651 to nucleotide 1940;
(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-1 deposited under accession number ATCC 209588;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 24 to amino acid 621;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid 621;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 encoding a protein having mocarhagin activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and
(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) the amino acid sequence of SEQ ID NO:8 from amino acid 24 to amino acid 439;
(c) the amino acid sequence of SEQ ID NO:8 from amino acid 192 to amino acid 439;
(d) fragments of the amino acid sequence of SEQ ID NO:8 encoding a protein having mocarhagin activity; and
(e) the amino acid sequence encoded by the cDNA insert of clone NMM-2 deposited under accession number ATCC 209589;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 85 to nucleotide 1401;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 154 to nucleotide 1401;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 658 to nucleotide 1401;
(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-2 deposited under accession number ATCC 209589;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 24 to amino acid 439;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 192 to amino acid 439;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 encoding a protein having mocarhagin activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and
(1) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 24 to amino acid 613;
(c) the amino acid sequence of SEQ ID NO:10 from amino acid 192 to amino acid 613;
(d) fragments of the amino acid sequence of SEQ ID NO:10 encoding a protein having mocarhagin activity; and
(e) the amino acid sequence encoded by the cDNA insert of clone NMM-9 deposited under accession number ATCC 209586;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 67 to nucleotide 1905;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 136 to nucleotide 1905;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 640 to nucleotide 1905;
(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-9 deposited under accession number ATCC 209586;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 24 to amino acid 613;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 192 to amino acid 613;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 encoding a protein having mocarhagin activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and
(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12;
(b) the amino acid sequence of SEQ ID NO:12 from amino acid 24 to amino acid 521;
(c) the amino acid sequence of SEQ ID NO:12 from amino acid 192 to amino acid 521;
(d) fragments of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity; and
(e) the amino acid sequence encoded by the cDNA insert of clone NMM-12 deposited under accession number ATCC 209585;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 78 to nucleotide 1640;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1640;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 651 to nucleotide 1640;
(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-12 deposited under accession number ATCC 209585;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 24 to amino acid 521;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 192 to amino acid 521;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and
(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:14;
(b) the amino acid sequence of SEQ ID NO:14 from amino acid 24 to amino acid 592;
(c) the amino acid sequence of SEQ ID NO:14 from amino acid 192 to amino acid 592;
(d) fragments of the amino acid sequence of SEQ ID NO:12 encoding a protein having mocarhagin activity; and
(e) the amino acid sequence encoded by the cDNA insert of clone NMM-13 deposited under accession number ATCC 209584;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 83 to nucleotide 1858;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 152 to nucleotide 1858;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 656 to nucleotide 1858;
(e) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-13 deposited under accession number ATCC 209584;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 24 to amino acid 592;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 192 to amino acid 592;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 encoding a protein having mocarhagin activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(k) a polynucleotide which encodes a species homologue of the protein of (f), (g) or (h) above; and
(l) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(h) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 62 to amino acid 462;
(c) fragments of the amino acid sequence of SEQ ID NO:16 encoding a protein having mocarhagin activity; and
(d) the amino acid sequence encoded by the cDNA insert of clone NMM-3 deposited under accession number ATCC 209587;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 3 to nucleotide 1388;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 186 to nucleotide 1388;
(d) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-3 deposited under accession number ATCC 209587;
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16 from amino acid 62 to amino acid 462;
(g) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 encoding a protein having mocarhagin activity;
(h) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(i) a polynucleotide which encodes a species homologue of the protein of (e) or (f) above; and
(j) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(g) above.
The present invention also provides a composition comprising a mocarhagin protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:18;
(b) the amino acid sequence of SEQ ID NO:18 from amino acid 197 to amino acid 621;
(c) fragments of the amino acid sequence of SEQ ID NO:18 encoding a protein having mocarhagin activity; and
(d) the amino acid sequence encoded by the cDNA insert of clone NMM-9ek deposited under accession number ATCC 209583;
the protein being substantially free from other mammalian proteins.
Yet other embodiments provide for a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 67 to nucleotide 1929;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 655 to nucleotide 1929;
(d) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone NMM-9ek deposited under accession number ATCC 209583;
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(f) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18 from amino acid 197 to amino acid 621;
(g) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 encoding a protein having mocarhagin activity;
(h) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(i) a polynucleotide which encodes a species homologue of the protein of (e) or (f) above; and
(j) a polynucleotide which hybridizes under stringent conditions to a polynucleotide of (a)-(g) above.
Compositions comprising an antibody which specifically reacts with the mocarhagin proteins or a fragments thereof having mocarhagin proteolytic activity are also provided.